Essential to oral biofilm development is the initial colonization by oral streptococci. The abundant oral streptococci prevent pathogens from arising in numbers that tips the balance to a pathogenic state. We have used an oral streptococcus Streptococcus parasanguinis as a model to study bacterial adhesion and colonization and identified a new family of bacterial serine-rich repeat protein (SRRP) named a fimbriae- associated protein-Fap1. Previous funding support has led to the discovery of a new glycosylation and secretion pathway responsible for biogenesis of Fap1. Biogenesis of Fap1 is controlled by a gene cluster encoding glycosyltransferases and accessory secretion proteins. Fap1-like SRRPs are highly conserved in Gram-positive bacteria and play an important role in bacterial colonization and virulence. We have generated genetic and biochemical tools and established Fap1 as a model system to investigate this important family of SRRP adhesins. In our study of Fap1 glycosylation, we have identified two putative glycosyltransferases (Gtf), Gtf1 and Gtf2 that are required for the first step of Fap1 glycosylation, as well as additional glycosyltransferases GalT1, GalT2 and Gly that are responsible for the subsequent Fap1 glycosylation steps. Gtf2 interacts with Gtf1 and functions as a molecular chaperone to enhance glycosyltransferase activity of Gtf1. GalT1 contains a novel domain of unknown function (DUF1792) that possesses glycosyltransferase activity. However the molecular events underlying how they contribute to Fap1 glycosylation is unknown. In our study of Fap1 secretion, we identified a protein complex consisting of two distinct glycosylation associated proteins, Gap1 and Gap3, which regulate Fap1 biogenesis. Gap3 is a lectin that organizes the accessory secretion protein complex. Gap1 is a putative glycosyltransferase that interacts with Gap3, and also stabilizes Gap3. However the biochemical function of the complex is unknown. Two specific aims are proposed: Aim 1 Dissect Fap1 glycosylation steps and determine the contribution of glycosyltransferases. We will determine A) how Gtf2 modulates Gtf1 by solving 3D structure of Gtf2 and define the underlying mechanism how Gtf2 modulates Gtf1; B) how DUF1972 functions as a new glycosyltransferase by solving its 3-D structure; C) how GalT2 and Gly contribute to Fap1 glycosylation. Aim 2 Define the role of the glycosylation-associated protein complex in Fap1 biogenesis. We will determine A) the role of Gap3 in Fap1 biogenesis as a lectin; B) the engagement of Gap1 in Fap1 biogenesis via three interactive mechanisms, 1) glycosylation of Gap3; 2) interaction with Gap3; and 3) prevention of Gap3 from proteolytic degradation since the presence of Gap1 stabilizes Gap3. As biogenesis of SRRPs is highly conserved, deciphering the molecular details that modulate bacterial adhesion by the Fap1 biogenesis will provide new information useful for the design of strategies to maintain the oral cavity healthy, as well as aid in the design of novel therapeutics that control pathogens that use SRRPs.